Infectious diseases are one of the leading causes of death worldwide. Emergence of antibiotic resistant bacteria causes serious concerns even for infections that once were treatable with antibiotics or other antimicrobial agents. A notable example is a recent outbreak of infections with methicillin resistant Staphylococcus aureus (MRSA). S. aureus infections were nosocomial and mostly confined to predisposed individuals. Originally, MRSA strains were found only in health care settings (HA-MRSA);however, more recently MRSA strains appear in the community (CA-MRSA) among individuals who bear no risk factor for the infections. In addition, increasing numbers of severe invasive infections with MRSA have been reported, which have resulted in and estimated 18,650 deaths during 2005 in the United States. Although vancomycin remains the last resort for treating MRSA infections, vancomycin resistant strains have begun to emerge among HA- and CA-MRSA isolates, necessitating the development of new strategies to combat MRSA infections. The antimicrobial substance C9ASA was identified from a natural isolate C9 of Bacillus pumilus. C9ASA is effective against some Gram-positive bacteria and exhibits the highest activity against S. aureus, including HA- and CA-MRSA, as well as Streptococcus pyogenes, a group A Streptococcus. Both S. aureus and group A Streptococcus are among the NIAID list of re-emerging pathogens. C9ASA is not produced by seven other B. pumilus strains thus far tested and is different from antibacterial substances that were previously isolated and reported from B. pumilus, suggesting that it is a novel compound. The long-term objectives of the proposed research are to develop a new drug to treat infections caused by MRSA and group A Streptococcus, and by using C9ASA and S. aureus as a model, to elucidate the mechanism by which Gram-positive pathogenic bacteria become resistant to certain antimicrobial agents. In Specific Aim 1, partially purified C9ASA will be further purified using HPLC and the molecular weight will be determined using tandem mass spectrometry. Proton NMR and multidimensional NMR will be applied to solve the structure of C9ASA. In Specific Aim 2, the mode-of-action of C9ASA will be determined by isolating and characterizing S. aureus mutants that are resistant to the substance. In addition, transcriptome analysis will be carried out to identify genes in S. aureus induced by C9ASA, which might uncover potential C9ASA targets. In Specific Aim 3, a genetic system for study of B. pumilus will be developed in order to facilitate experiments proposed in Specific Aim 4. Based on the hypothesis that B. pumilus lacks natural competence due to the absence of comS, a gene essential for B. subtilis competence, the comS gene will be introduced into B. pumilus C9 using a recently developed conjugative system to endow the C9 strain with natural competence. In Specific Aim 4, genes required for C9ASA production will be identified using mini-Tn10 mutagenesis. These genes likely include biosynthesis genes and regulatory genes involved in C9ASA production. PUBLIC HEALTH RELEVANCE: Invasive MRSA infections are a serious threat to public health worldwide. This proposal describes studies aimed at characterizing what might be a promising anti-MRSA substance for combating this re-emerging infectious threat.